The specific activation (and repression) of the erythroid transcriptome is the critical feature of erythropoiesis. While a great deal is known about the genetic and epigenetic control of the globin loci, much less is known about the regulation of other erythroid genes. The goal of this project is to integrate our knowledge about epigenetic changes with RNA analysis to test the hypothesis that epigenetic changes are responsible for changes in gene expression in differentiating hematopoietic cells. Our previous studies focused on point mutations that identified critical regulatory elements in specific genes (Laflamme K, Owen AN, Devlin EE, Yang MQ, Wong C, Steiner LA, Garrett LJ, Elnitski L, Gallagher PG, Bodine DM. Functional analysis of a novel cis-acting regulatory region within the human ankyrin gene (ANK-1) promoter. Mol Cell Biol. 30: 3493-502, 2010; Yang MQ, Laflamme K, Gotea V, Joiner CH, Seidel NE, Wong C, Petrykowska HM, Lichtenberg J, Lee S, Welch L, Gallagher PG, Bodine DM, Elnitski L. Genome-wide detection of a TFIID localization element from an initial human disease mutation. Nucleic Acids Res. 39 (6): 2175-87, 2011; and Gallagher PG, Steiner LA, Liem RI, Owen AN, Cline AP, Seidel NE, Garrett LJ, Bodine DM. Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis. J Clin Invest. 120 (12): 4453-65, 2010). We recently demonstrated that a concert of local epigenetic changes coupled with long range interactions were responsible for activation of the ANK1 gene in erythroid cells (Yocum AO, Steiner LA, Seidel NE, Cline AP, Rout ED, Lin JY, Wong C, Garrett LJ, Gallagher PG, Bodine DM. A Tissue Specific Chromatin Loop Activates the Erythroid Ankyrin-1 Promoter. Blood. 120:3586-93, 2012). We are now moving on to genome-wide analyses. We have completed RNA-Seq analyses of erythroblasts, megakaryocytes and MEP. Our findings include the identification of over 600 novel long non-coding RNAs, many of which are expressed specifically in one of the three cell types. We plan to extend these analyses by correlating the changes in gene expression we observe with the changes in the epigenetic profile of the genome that we are developing in a different project. This will allow for the first time direct comparisons of gene expression and epigenetic signatures in primary cells.